PROJECT SUMMARY Exposure to perfluoroalkyl substances (PFASs) is ubiquitous in the U.S. and worldwide. A breadth of data from animal studies show effects of prenatal and postnatal PFAS exposures on multiple tissues, accompanied by systemic biological responses and altered levels of lipids, glucose, inflammatory markers and metabolism- regulating hormones in peripheral blood. In line with the experimental evidence, our previous prospective investigations showed apparent effects of prenatal PFAS exposures on adverse metabolic and immune outcomes in children, as well as associations of adult PFAS exposures with higher risks of gestational and type 2 diabetes (T2D). As dose-dependence and mechanisms of potential PFAS toxicity in humans are not yet understood, an urgent need exists to elucidate associated health risks and implicated mechanisms. Moreover, although PFASs persist in blood and tissues for years, previous birth cohort research has focused almost exclusively on short-term impacts of prenatal exposure. Therefore, we propose an innovative life-course epidemiologic design that integrates lifetime PFAS exposures and untargeted high-resolution metabolomics (HRM). This design will allow for the identification of biological pathways potentially altered by early-life PFAS exposures and critical periods of susceptibility (Aim 1) as well as of pathways associated with changes in clinical cardiometabolic markers in young adults (Aim 2). Gaining insight from the first two aims, we will then be able to quantify the relative contribution of identified pathways in the PFAS-associated cardiometabolic disease pathogenesis (exploratory Aim 3). To pursue these aims, we will leverage the unique resource of a well- characterized prospective Faroese cohort of 1,022 individuals born in 1986/7 who has now reached adulthood, and utilize state-of-the-art HRM methodologies that offer a wide coverage of biological pathways, including those suspected to mediate PFAS metabolic effects. Five major PFASs have already been measured in whole cord blood and participants' serum at ages 7, 14, 22 and 28 years. Clinical assessments in adulthood included anthropometry, blood pressure and 2 hour-oral glucose tolerance tests, complimenting available obesity measures from childhood examinations. The proposed work will utilize archived fasting plasma from 500 cohort participants for HRM analyses at ages 22 and 28, thereby offering a unique opportunity to capture both long- term and late-onset effects of early-life PFAS exposures. Findings from this exploratory R21 research will provide new insights into systemic biological responses to PFAS exposures and potential critical periods, while also having the potential to identify novel mechanistic pathways for obesity, T2D and heart disease at an early adulthood age-window, when interventions are effective and can benefit the population.